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Abstract：
Many biomolecular condensates are enriched in and depend on RNAs and RNA binding proteins (RBPs). So far, only a few studies have addressed the characterization of the intermolecular interactions responsible for liquid-liquid phase separation (LLPS) and the impact of condensation on RBPs and RNAs. Here, we present an approach to study protein-RNA interactions inside biomolecular condensates by applying cross-linking of isotope labeled RNA and tandem mass spectrometry to phase-separating systems (LLPS-CLIR-MS). LLPS-CLIR-MS enables the characterization of intermolecular interactions present within biomolecular condensates at residue-specific resolution and allows a comparison with the same complexes in the dispersed phase. We observe that sequence-specific RBP-RNA interactions present in the dispersed phase are generally maintained inside condensates. In addition, LLPS-CLIR-MS identifies structural alterations at the protein-RNA interfaces, including additional unspecific contacts in the condensed phase. Our approach offers a procedure to derive structural information of protein-RNA complexes within biomolecular condensates that could be critical for integrative structural modeling of ribonucleoproteins (RNPs) in this form.
摘要：
许多生物分子缩合物富含并依赖于RNA和RNA结合蛋白(RBP)。到目前为止,只有少数研究解决了负责液-液相分离（LLPS）的分子间相互作用的表征以及缩合对RBP和RNA的影响。这里,我们提出了一种通过将同位素标记的RNA的交联和串联质谱应用于相分离系统（LLPS-CLIR-MS）来研究生物分子缩合物中蛋白质-RNA相互作用的方法。LLPS-CLIR-MS能够以残基特异性分辨率表征生物分子缩合物中存在的分子间相互作用，并允许与分散相中的相同复合物进行比较。我们观察到存在于分散相中的序列特异性RBP-RNA相互作用通常保持在缩合物内。此外,LLPS-CLIR-MS鉴定蛋白质-RNA界面的结构改变，包括冷凝相中的额外非特异性接触。我们的方法提供了一种程序来获取生物分子缩合物中蛋白质-RNA复合物的结构信息，这对于这种形式的核糖核蛋白（RNP）的整合结构建模至关重要。
